Materials

The plasmid DNA pCMV6-AC-GFP was purchased from OriGene (PS100010, Rockville, US). HindIII (R3104) and NotI (R3189) restriction enzymes were obtained from New England Biolabs, Inc. (Ipswich, US). The cell culture media, including RPMI1640 (SH30027.01) and Dulbecco’s Modified Eagle Medium (DMEM, SH30243.01) were purchased from Cytiva (Hyclone™, Marlborough, US). Fetal bovine serum (10,091,148, Gibco™), the antibiotic Penicillin–Streptomycin (15,140,122, Gibco™), protease inhibitor cocktail (78,441), dispase II (17,105,041, Gibco™), anti-human EGFR antibody (AHR5062, clone R19/48, Invitrogen™), Alexa Fluor™ 594 conjugated goat anti-mouse IgG (A-11005), Alexa Fluor™ 488 conjugated goat anti-mouse IgG (A-11029), Foxp3/Transcription Factor Staining Buffer Set kit (00–5523-00) were supplied by Thermo Fisher (Waltham, US). RIPA buffer (P0013C), biotinylated goat anti-mouse IgG (H + L) (A0286) and horseradish peroxidase conjugated streptavidin (A0303) were obtained from Beyotime (Jiangsu, China). The primary antibody anti-tubulin was purchased from Cowin Biotech (CW0098, Jiangsu, China). The CytoTox 96® Non-Radioactive Cytotoxicity Assay kit was purchased from Promega (G1780, Madison, US). DNase I was obtained from Roche (10,104,159,001, Basel, Switzerland). The Mouse 1 × Lymphocyte Separation Medium was purchased from Dakewe Biotech Co., Ltd. (7,211,011, Guangdong, China). Mitomycin C was obtained from Selleck (S8146, Houston, US). Murine monoclonal antibodies, including anti-CD45 (103,125, 30-F11, Pacific Blue™), anti-CD3 (100,220, 17A2, PE/Cyanine7), anti-CD4 (100,405, GK1.5, FITC), anti-CD4 (100,527, RM4-5, PE/Cyanine7), anti-CD25 (102,007, PC61, PE), anti-Foxp3 (126,407, MF-14, Alexa Fluor® 647), anti-CD8α (100,712, 53–6.7, APC), anti-PD-1 (135,207, 29F.1A12, PerCP/Cyanine5.5), anti-IFN-γ (505,808, XMG1.2, PE), anti-CD11b (101,205, M1/70, FITC), anti-Gr-1 (108,408, RB6-8C5, PE), were purchased from BioLegend (San Diego, US). Brefeldin A (420,601), CFSE (carboxyfluorescein diacetate succinimidyl ester, 65–0850-84) and the purified anti-mouse CD16/32 antibody (101,302) were also supplied by BioLegend. Goat anti-mouse IgG antibody (HRP) polymer (PV-9002) and DAB (ZLI-9017) were obtained from ZSGB-BIO (Beijing, China). ELISA Coating Buffer (421,701) and ELISA Assay Diluent (421,203) were purchased for BioLegend. Human EGFRvIII Protein was obtained from ACRO (EGI-H52H4, Newark, US). A 1.25% solution of tribromoethanol was purchased from Nanjing Aibei Biotechnology Co., Ltd (M2920, Jiangsu, China). Geneticin (G418) was obtained from Sangon Biotech (A100859, Shanghai, China). DOTAP (1,2-dioleoyl-3-trimethylammonium-propane) was purchased from Avanti Polar Lipids, Inc. (890,890, Alabaster, US), and 2-mercaptoethanol was obtained from Macklin Inc. (M6230, Shanghai, China). The nitrocellulose membrane was purchased from Bio-Rad Laboratories, Inc. (1,620,115, Hercules, US).